Quantitative Fluorescence Assays Using a Self

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Quantitative Fluorescence Assays Using a Self-Powered
Paper-Based Microfluidic Device and a Camera-Equipped
Cellular Phone
Nicole K. Thom, Gregory G. Lewis, Kimy Yeung and Scott T. Phillips*
Department of Chemistry, The Pennsylvania State University, University Park,
Pennsylvania 16802, United States
*Corresponding author E-mail: sphillips@psu.edu
Supporting Information
Table of Contents
Materials used
Fluorescence Assay
Patterning paper and tape layers
Assembly of the device
Characterization of the Battery
Measuring current and potential of the battery
Measuring LED output over time
Fluorescein Calibration Curve
Making buffer
Making fluorescein solutions
Cell phone case design
Making the calibration curve with fluorescein solutions
Assay for β-D-galactosidase
Synthesis of the assay reagent
Running an Assay
Running multiple assays in the same device
References
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Materials Used
Tape used was Ace Hardware brand plastic carpet tape (product # 50106). The paper was Whatman
Chromatography Paper #1. Sodium nitrate, silver nitrate, aluminum chloride, silver sheet, aluminum
sheet, β-D-galactosidase, fluorescein, and mono-and di-basic sodium phosphate were all purchased
commercially and used without further purification. The filter used is a Kodak Wratten 2 filter No.
12, purchased from Edmond Optics (product # NT45-467). The LED was purchased from DigiKey
(product # P14150CT-ND). The cell phone case is the Metallic Slider Case (steel) for iPhone 4S
made by Incase Designs Corp.
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Fluorescence Assay
Figure S1. Layers of the assay device with dimensions labeled. The values are all listed in mm.
The half of layers 5–9, which is constantly, tape, is the assay side of the device, and the other
side (which alternates between paper and tape) is the battery side of the device.
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Figure S1 Continued. Layers of the assay device with dimensions labeled. The values are
all listed in mm. The half of layers 5–9, which is constantly, tape, is the assay side of the device,
and the other side (which alternates between paper and tape) is the battery side of the device.
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Patterning paper and tape layers
Patterning the paper and tape layers has been reported previously by Noh et al.1
Assembly of the device
The wax on the paper layers was melted for 105 s on a 150° C hot plate with a stack of
papers on top of it. After cooling, the battery side of layer 7 had salt added to it, 0.75 µL of saturated
NaNO3 solution was added in two aliquots, drying under vacuum for 30 min after each addition. The
battery side of layer 5 had 0.25 µL of the appropriate metal solution placed in each spot. The metal
solutions used were 1 M AlCl3 and 3 M AgNO3. The placement of the salts can be seen in Figure S1.
After the addition of the salts, layer 5 was dried under vacuum for 30-60 min.
The protective layer on one side of layer 10 was removed, and layer 10 was added to layer
11, with the adhesive attaching them together. Layer 11 was then cut out from the surrounding paper,
with scissors, using layer 10 as a guide. The protective layer on one side of layer 12 was removed
and layer 12 was adhered to layer 11.
The battery side of layer 8 had one protective cover removed, and it was adhered to the
battery side on layer 9. These were then cut out, with scissors, using layer 8 as a guide. The
remaining protective sheet on layer 10 was then removed, the holes were filled with technicloth discs
of the appropriate sizes, and layer 9 was attached.
The battery side on layer 6 then had one protective sheet removed and was attached to the
battery side on layer 7 (already containing the salts). These are then cut out, with scissors, using layer
6 as a guide. The remaining protective sheet on layer 8 was then removed, technicloth discs of the
appropriate sizes are placed in the holes, and layer 7 was attached.
The inner edge on the battery side of layer 5 was cut out, making sure that it is short enough
to not overlap with the assay side of the layer. A protective sheet on layer 4 was then removed, and
layer 4 was attached to layer 5. The assay sides of layers 5–9 were all stacked onto layer 4. Then the
layer 4 stack was attached to the stacked layers for the bottom of the device, with technicloth discs
placed in the holes in layer 6. A piece of black electrical tape was cut and placed at the bottom of the
assay region, and the entire stack is placed under a 4.6 kg weight for at least 15 minutes (but not
longer than 2 days) before continuing.
The 10 copies of layer 3 were then stacked together, and set aside. Layer 2 was attached to
layer 1, and the pieces were then cut out, with scissors, and this was set aside. Clear plastic straws
were cut into 2 mm tall cylinders using a razor, and these were set aside.
The protective layer on layer 4 was removed and pieces of Al and Ag are placed in the line of holes
further from the assay side of the device. Each piece of metal had a small amount of gallium placed
on top, and a piece of Cu tape was placed down connecting all 6 pieces of metal. A thin strip of
carpet tape was then cut and placed over the copper tape, and the protective layer on the carpet tape
was removed.
The Ag pieces were then placed in the remaining Ag holes, a small amount of gallium was
placed on each metal piece, and a strip of Cu tape was placed connecting all three pieces. This was
repeated for the Al pieces in the Al holes. A thin piece of carpet tape is placed in between the two
pieces of Cu tape, above where the LED will be placed, and the protective sheet is removed.
The stack of layer 3 was then attached to the top of layer 4. The LED was placed, with the
ground on the Al side of the copper tape, and a small amount of gallium was used to attach the LED
to the separate pieces of Cu tape. One piece of straw is placed in the assay region, directly in front of
the LED. The battery input region hole is then filled with technicloth discs and covered with layers 1
and 2.
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Characterization of the Battery
Measuring current and potential of the battery
To measure the current and potential of this battery, the device was built as described above, without
including layer 3, the LED, or the piece of straw. Alligator clips were used to attached the pieces of
copper tape to a multimeter, which was used to monitor either current or potential. The battery was
run with 80 µL deionized water.
Table S1. Data collected for the current and potential of the battery.
min
Time
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
11.5
12
12.5
13
13.5
14
14.5
15
15.5
16
mA
Trial 1
0
0
0
0
0
0
0
0
0
0
0
0.07
0.28
0.39
0.58
0.61
0.63
0.64
0.65
0.65
0.64
0.58
0.55
0.49
0.41
0.31
0.17
0.11
0.11
0.1
0.09
-
mA
Trial 2
0
0
0
0
0
0
0
0
0.05
0.24
0.39
0.52
0.6
0.64
0.64
0.63
0.64
0.65
0.61
0.55
0.46
0.4
0.37
0.27
0.25
0.22
0.19
0.13
0.12
0.12
0.11
0.1
0.1
mA
Trial 3
0
0
0
0
0
0
0
0
0
0.01
0.09
0.22
0.4
0.53
0.6
0.67
0.68
0.67
0.65
0.62
0.62
0.52
0.58
0.5
0.39
0.35
0.29
0.26
0.25
0.23
0.28
0.26
0.17
V
Trial 4
0
0.02
0.02
0.04
0.04
0.05
0.72
1.75
2.58
2.74
2.8
2.86
2.87
2.84
2.88
2.91
2.92
2.92
-
V
Trial 5
0
0.02
0.02
0.03
0.03
0.04
0.06
0.92
2.22
2.65
2.69
2.68
2.66
2.67
2.68
2.68
2.7
2.68
-
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Table S1 Continued. Data collected for the current and potential of the battery.
min
Time
16.5
17
17.5
18
mA
Trial 1
-
mA
Trial 2
0.1
0.09
-
mA
Trial 3
0.1
0.12
0.1
0.18
V
Trial 4
-
V
Trial 5
-
Measuring LED output over time
The LED output over time was measured using the same fluorescence device described above, with a
few small changes. Layers 1 and 2 were extended the whole length of the device, and a small hole
(1.5 mm diameter) centered over the straw. A variable photoresistor had a small ring of tape placed
on the edge of it, and it was centered and taped down onto the hole over the assay region. The battery
was started with 80 µL deionized water and the resistance was measured over time.
Table S2. Data collected for LED output over time.
min
Time
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
kΩ
Trial 1
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2000
2000
646
609
578
540
515
503
490
kΩ
Trial 2
2015
2015
2015
2015
2015
2015
2015
1049
467
267
221
210
204
215
218
222
225
229
231
234
238
225
233
kΩ
Trial 3
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
1580
930
740
620
535
420
395
375
355
kΩ
Trial 4
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
1200
569
405
285
240
215
197
185
175
166
160
156
152
kΩ
Trial 5
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
2015
1760
878
512
440
385
345
308
283
265
253
242
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Table S2 Continued. Data collected for LED output over time.
min
Time
11.5
12
12.5
13
13.5
14
14.5
15
15.5
16
16.5
17
17.5
18
18.5
19
19.5
20
20.5
21
21.5
22
22.5
23
23.5
24
24.5
25
25.5
26
26.5
27
27.5
28
28.5
29
29.5
30
kΩ
Trial 1
475
452
434
420
412
407
399
392
383
373
368
366
365
364
362
361
360
361
359
360
363
367
368
371
378
378
382
387
393
400
408
417
429
440
454
466
480
489
kΩ
Trial 2
238
238
240
243
261
249
250
271
271
255
254
258
283
270
269
273
276
282
286
290
295
299
304
316
330
341
360
379
413
433
450
450
457
477
500
536
555
kΩ
Trial 3
335
320
309
298
290
278
270
262
258
254
250
245
243
240
238
235
234
233
233
233
233
233
235
236
237
240
243
245
247
248
252
257
260
266
270
277
281
290
kΩ
Trial 4
150
147
145
143
142
141
141
141
142
143
143
144
144
144
145
146
147
148
150
151
152
153
154
154
154
154
155
156
158
159
160
161
163
165
166
169
171
173
kΩ
Trial 5
233
225
222
217
213
210
208
205
203
202
201
199
198
199
200
200
200
202
202
203
203
203
203
204
203
206
207
207
209
211
212
214
217
218
221
224
227
230
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Fluorescein calibration curve
Making buffer
1 L pH 7.6 sodium phosphate buffer was made by dissolving 1.79 g (13 mmol) monobasic sodium
phosphate monohydrate and 12.35 g (87 mmol) dibasic sodium phosphate anhydrous in deionized
water. The pH of this solution was found to be 7.61.
Making fluorescein solutions
11.6 mg (0.035 mmol) fluorescein free acid was dissolved in 500 mL of the buffer solution, giving a
69.8 µM stock solution. The stock solution was serially diluted with the buffer solution to give the
tested concentrations: 5.93 µM, 5.00 µM, 4.00 µM, 3.00 µM, 2.00 µM, 1.00 µM, 0.50 µM and 0.25
µM fluorescein in sodium phosphate buffer.
Cell phone case design
The cell phone case (Figure S2) was designed to place the assay at the nearest focal point of the
camera while blocking out all external light. The case was purchased commercially, and a black
polyethylene tube (cut to 2”) was glued onto the case over the cutout for the camera. Two caps were
designed for used on the end of the tube. One contained a filter (Kodak Wratten 2 No. 12) to remove
excess LED light from the picture (Figure S3); the second cap was used to keep the focal length the
same for pictures with and without the filter.
Figure S2. Picture of the cell phone case put together and of the various pieces, showing how
it is put together. (a) The cell phone case and black tube as purchased. (b) the tube cut to 2” long
and glued onto the cell phone case. The white end on the tube is a piece of tape. (c) The tube
glued onto the cell phone case. (d) The two vial lids that are used to make a cap for the end of the
tube. (e) Both vial lids with the septa removed. The black lid has a tape ring on the bottom and
top, and the blue lid has a ring of tape on top holding on the yellow filter, and a second ring of
tape on the other side of the filter. (f) The two lids stacked to form the cap. The blue lid is taped
into the black lid. (g) The cap placed onto the cell phone case with the tube. A single protective
layer from the tape remains between the cap and the tube, allowing the cap to be adhered for
pictures, but removable to switch to the cap without the filter. The ring of tape on the top of the
cap also contains a protective layer. This is used to adhere the cap to the fluorescence device to
block out all light when taking a picture, and the protective layer allows for easy removal from
the device. (h) a second angle of the device in (g).
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Figure S3. Graph showing the range of wavelengths for each component in the assay. This
shows that the filter used is able to block out most of the background LED light, while allowing
through most of the emission light. The blue data is the LED emission, the green data is the
fluorescein emission, and the black line is the transmission through the filter.
Making the calibration curve with fluorescein solutions
To run an assay, 80 µL deionized water is spotted on the battery side of the device, and 70 µL of the
sample is spotted in the assay region. The device was then connected to the case without a filter, and
after 16-17 min a picture was taken, the case was then switched to containing a filter and a second
picture was taken. This as repeated 3-4 times for each concentration of fluorescein solution tested.
Table S3. Data collected for the calibration curve of buffered fluorescein using different LEDs for
every trial, without using a filter or the cell phone case. All collected values are median value using
the green channel of the histogram function in Adobe Photoshop.
Concentration
(µM)
Trial 1
Trial 2
Trial 3
Trial 4
0
31
31
10
10
1
18
19
37
38
1.5
47
59
30
30
2.5
61
55
23
24
5
80
87
117
120
7.5
53
48
151
58
10
88
92
51
93
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Figure S4. Graph showing the relative improvement in reproducibility for measuring
fluorescein from the implementation of the various features of the device. The blue data is from
devices imaged with a cell phone camera only, with no case, filter, or alignment circles used. The
black data is from devices imaged using a cell phone camera as described above.
Table S4. Data collected for the calibration curve of buffered fluorescein using different LEDs for
every trial, without using a filter. All collected values are median value using the green channel of
the histogram function in Adobe Photoshop.
Concentration
(µM)
Trial 1
Trial 2
Trial 3
Trial 4
0
17
13
0.25
12
14
20
0.5
28
37
14
1
72
86
45
49
2
93
19
29
3
57
70
33
4
121
65
5
82
150
170
104
Table S5. Data collected for the calibration curve of buffered fluorescein using different LEDs for
every trial, using a filter. All collected values are median value using the green channel of the
histogram function in Adobe Photoshop.
Concentration
(µM)
Trial 1
Trial 2
Trial 3
Trial 4
0
5
5
0.25
6
5
7
0.5
8.5
10
5
1
25.5
30
10
16
2
33
16
10
3
27
31
17
4
54
29
5
51
66
84
50
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Table S6. Data collected for the calibration curve of buffered fluorescein using the same LEDs for
every trial, without using a filter. All collected values are median value using the green channel of
the histogram function in Adobe Photoshop.
Concentration
(µM)
Trial 1
Trial 2
Trial 3
0.25
3
0.5
4
1
22
21
25
2
25
41
35
3
71
80
52
4
77
96
73
5
80
76
100
5.93
52
96
75
Table S7. Data collected for the calibration curve of buffered fluorescein using different LEDs for
every trial, using a filter. All collected values are median value using the green channel of the
histogram function in Adobe Photoshop.
Concentration
(µM)
Trial 1
Trial 2
Trial 3
0.25
3
0.5
4
1
8
10
9
2
13
16
14
3
37
42
35
4
43
51
47
5
35
46
37
5.93
27
54
42
Assay for β-D-galactosidase
Synthesis of the assay reagent
General Experimental Procedures
All reactions that required anhydrous conditions were performed in flame-dried glassware under
a positive pressure of argon. Air- and moisture-sensitive liquids were transferred by syringe or
stainless steel cannula. Organic solutions were concentrated by rotary evaporation (25–40
mmHg) at ambient temperature, unless otherwise noted. Thin layer chromatography was carried
out on Dynamic Adsorbants silica gel TLC (20Å~20 cm w/h, F-254, 250 μm).
Instrumentation
Proton nuclear magnetic resonance (1H-NMR) spectra and carbon nuclear magnetic resonance
spectra (13C-NMR) were recorded using a Bruker CDPX-300 (300 MHz) or AV-360 (360 MHz)
at 25 °C. Proton chemical shifts are expressed in parts per million (ppm, δ scale) and are
referenced to methanol (CD3OD, 3.31 ppm). Data are represented as follows: chemical shift,
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multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and/or multiple
resonances, br s = broad singlet, dd = doublet of doublet), integration, and coupling constant (J)
in Hertz. Carbon chemical shifts are expressed in parts per million (ppm, δ scale) and are
referenced to methanol (CD3OD, 49.0 ppm).
Scheme 1. Synthesis of reagent 1.
Methyl 2-(3-oxo-6-(4-((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2Hpyran-2-yloxy)benzyloxy)-3H-xanthen-9-yl)benzoate (5):
Compound 32 (0.20 g, 0.44 mmol, 1.0 equiv) was dissolved in dry tetrahydrofuran (4.4 mL) and
the solution was cooled to 0 °C. Phosphorous tribromide (1.0 M solution in dichloromethane,
180 L, 0.18 mmol, 0.4 equiv) was added dropwise, and the solution was stirred at 0 °C for 20
min. The reaction was diluted with dichloromethane (10 mL) and quenched with saturated
sodium bicarbonate (20 mL). The organic layer was collected, and the aqueous layer was
extracted with dichloromethane (3 × 20 mL). The combined organic layers was dried over
sodium sulfate, filtered, and concentrated by rotary evaporation, yielding a white solid. The
white solid was used without further purification.
The white solid (0.13 g, 0.26 mmol, 1.0 equiv), compound 43 (0.09 g, 0.26 mmol, 1.0 equiv), and
potassium carbonate (0.04 g, 0.29 mmol, 1.1 equiv) were dissolved in dry dimethylformamide (3
mL). The solution was heated at 50 °C for 19 h. The reaction mixture was cooled to room
temperature and the solvent was removed by rotary evaporation. The residue was purified by
preparative HPLC to afford compound 5 as an orange solid (52 mg, 85 µmol, 33%). IR (cm-1):
3328.9, 2923.2, 1723.2, 1589.9; 1H-NMR (300 MHz, CD3OD): δ 8.31 (dd, 1 H, J = 2.07, 9.19),
7.87 – 7.75 (m, 2 H), 7.44 – 7.41 (m, 1 H), 7.42 (d, 2 H, J = 10.44), 7.29 (d, 1 H, J = 2.71), 7.16
(d, 2 H, J = 10.48), 7.05 – 6.95 (m, 3 H), 6.59 (dd, 1 H, J = 2.45, 11.53), 6.49 (d, 1 H, J = 2.46),
5.21 (s, 2 H), 3.91 (d, 1 H, J = 3.5), 3.83 – 3.67 (m, 5 H), 3.61 (s, 3 H), 3.61 (dd, 1 H, J = 6.46,
14.12); 13C-NMR (360 MHz, CD3OD): δ 187.2, 167.0, 166.0, 161.5, 159.3, 156.3, 135.5, 134.1,
132.6, 132.2, 131.8, 131.5, 131.3, 131.0, 130.7, 130.5, 129.3, 118.1, 117.9, 116.3, 105.5, 102.9,
102.5, 77.0, 74.9, 72.3, 71.8, 70.2, 62.4, 52.9; MS (TOF MS AP+, m/z): 615.1 (M + 1); HRMS
(TOF MS ES+) Calculated for C34H31O11 (M + 1): 615.1866; Found 615.1862.
2-(3-Oxo-6-(4-((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran2-yloxy)benzyloxy)-3H-xanthen-9-yl)benzoic acid (1):
Compound 5 was dissolved in dry methanol (950 µL) and tetrahydrofuran (380 µL), and the
solution was stirred at room temperature. To the stirring solution was added a solution of
LiOHH2O in deionized water (6 mg, 0.14 mmol, 180 L). The reaction mixture was stirred at
room temperature for 4 h and an additional portion of LiOHH2O in deionized water (6 mg, 0.14
mmol, 180 L) was added. After 17 h, the mixture was diluted with water and carefully
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acidified using Dowex® C-211 H+ exchange resin (final pH = 5). The resin was removed by
filtration, and the filtrate was concentrated under reduced pressure, yielding a yellow residue.
The residue was purified by preparative HPLC to afford compound 1 as a yellow solid (21 mg,
35 mol, 49%). IR (cm-1): 3343.5, 2924.2, 1737.4; 1H-NMR (360 MHz, CD3OD): δ 8.03 (d, 1
H, J = 6.88), 7.75 – 7.66 (m, 2 H), 7.39 (d, 2 H, J = 8.68), 7.20 (d, 2 H, J = 6.81), 7.14 (d, 2 H, J
= 8.72), 6.95 (s, 1 H), 6.75 (s, 2 H), 6.69 – 6.65 (m, 2 H), 6.55 (dd, 1 H, J = 2.38, 8.86), 5.09 (s, 2
H), 3.91 (d, 1 H, J = 3.21), 3.82 – 3.67 (m, 5 H), 3.60 (dd, 1 H, J = 3.39, 9.76); 13C-NMR (360
MHz, CD3OD): δ 171.9, 162.7, 159.1, 155.4,, 154.3, 152.8, 135.5, 131.7, 131.0, 130.7, 130.3,
130.2, 126.7, 126.2, 117.8, 113.9, 113.4, 112.4, 103.8, 102.9, 77.0, 74.8, 72.3, 71.1, 70.2, 62.4;
MS (TOF MS AP+, m/z): 601.1 (M + 1); MS (TOF MS AP-, m/z): 599.1 (M – 1); HRMS (TOF
MS ES+) Calculated for C33H29O11 (M + 1): 601.1710; Found 601.1709.
Running an assay
A stock solution of 1 was made in phosphate buffer (200 mM, pH 7.6) and used for all β-Dgalactosidase assays. Added to 900 µL of the stock solution was 100 µL of β-D-galactosidase in
200 mM phosphate buffer, pH 7.6. The solution was mixed for 30 minutes before 75 µL of the
sample was added to the sample holder of the device. After 15 minutes of mixing, 90µL of
deionized water was added to the battery portion of the device to turn on the LED. After the
sample was added to the device, the device was connected to the case, containing the filter, and
the sample was imaged using an iPhone 4S. The images were analyzed using Adobe Photoshop,
measuring the median green value for each sample using the histogram function.
Table S8. Data for the calibration curve for β-D-galactosidase. All collected values are median
value using the green channel of the histogram function in Adobe Photoshop.
Concentration
(nM)
0
1
2
3
4
5
6
8
10
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Trial 6
40
39
38
38
34
34
48
49
52
50
53
53
69
71
62
63
51
51
73
75
73
75
70
72
82
85
75
78
78
82
94
97
96
98
88
91
92
95
95
98
90
93
98
101
98
101
98
102
106
109
105
109
103
108
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This journal is © The Royal Society of Chemistry 2013
Table S8 Continued. Data for the calibration curve for β-D-galactosidase. All collected
values are median value using the green channel of the histogram function in Adobe Photoshop.
12
14
15
16
18
20
115
120
115
119
106
110
110
117
109
115
115
119
113
116
106
109
108
111
108
113
109
114
114
119
110
114
112
115
109
114
111
117
112
119
108
111
Running multiple assays in the same device
To run multiple assays in the same microfluidic device, the first assay solution is pipetted into the
assay region, and pictures are taken, then the assay solution is removed. The assay region is rinsed
with the next assay solution three times (by pipetting in and out the new solution), then the next assay
solution is pipetted into the assay region and pictures are taken. This process is continued as long as
the battery time is between 15 and 22 minutes.
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This journal is © The Royal Society of Chemistry 2013
Copies of NMR spectra
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Electronic Supplementary Material (ESI) for RSC Advances
This journal is © The Royal Society of Chemistry 2013
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This journal is © The Royal Society of Chemistry 2013
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This journal is © The Royal Society of Chemistry 2013
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This journal is © The Royal Society of Chemistry 2013
References
1. H. Noh, S. T. Phillips, Anal. Chem., 2010, 82, 8071.
2. A. K. Ghosh, S. Khan, D. Farquhar. Chem. Commun., 1999, 24, 2527.
3. M. Adamczyk, J. Grote, J. A. Moore. Bioconjugate Chem., 1999, 10, 544.
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